Dopaminergic Cell Line Research Articles

Mitochondrial‐Targeting Vitamin B3 Ameliorates the Phenotypes of Parkinson's Disease In Vitro and In Vivo by Restoring Mitochondrial Function

AbstractParkinson's disease (PD) is the second most common neurodegenerative disease and is characterized by progressive dopaminergic neuron loss, which affects millions of people worldwide, especially elderly individuals. Mitochondrial dysfunction is believed to be one crucial causative factor of PD. Therefore, restoring mitochondrial function is highly recommended for the treatment of PD. Herein, it is found that vitamin B3 (VB3) protects the dopaminergic cell line SH‐SY5Y against rotenone‐induced apoptosis by restoring mitochondrial dysfunction. To further improve the efficacy, a derivative of VB3, mitochondrial‐targeting VB3 (Mito‐VB3), is developed. It is demonstrated that Mito‐VB3 rescues SH‐SY5Y cells from the neurotoxicity of oxidative stress. More importantly, Mito‐VB3 ameliorates the phenotypes of the Drosophila PD model. Mechanistically, Mito‐VB3 enhances nicotinamide adenine dinucleotide (NAD+) levels, reduces ROS generation, elevates ATP levels, and restores mitochondrial membrane potential. Meanwhile, Mito‐VB3 promotes mitochondrial biogenesis by activating the NAD+/SIRT1/PGC1α pathway. In summary, the results demonstrate that Mito‐VB3 displays remarkable neuroprotective effects in the cell and Drosophila PD models and that Mito‐VB3 might serve as a potential therapeutic candidate for the treatment of PD and other mitochondrial dysfunction‐related diseases.

Neuroprotective Effects of a Novel Demeclocycline Derivative Lacking Antibiotic Activity: From a Hit to a Promising Lead Compound.

The antibiotic tetracycline demeclocycline (DMC) was recently reported to rescue α-synuclein (α-Syn) fibril-induced pathology. However, the antimicrobial activity of DMC precludes its potential use in long-term neuroprotective treatments. Here, we synthesized a doubly reduced DMC (DDMC) derivative with residual antibiotic activity and improved neuroprotective effects. The molecule was obtained by removal the dimethylamino substituent at position 4 and the reduction of the hydroxyl group at position 12a on ring A of DMC. The modifications strongly diminished its antibiotic activity against Gram-positive and Gram-negative bacteria. Moreover, this compound preserved the low toxicity of DMC in dopaminergic cell lines while improving its ability to interfere with α-Syn amyloid-like aggregation, showing the highest effectiveness of all tetracyclines tested. Likewise, DDMC demonstrated the ability to reduce seeding induced by the exogenous addition of α-Syn preformed fibrils (α-SynPFF) in biophysical assays and in a SH-SY5Y-α-Syn-tRFP cell model. In addition, DDMC rendered α-SynPFF less inflammogenic. Our results suggest that DDMC may be a promising drug candidate for hit-to-lead development and preclinical studies in Parkinson’s disease and other synucleinopathies.

Protective effects of cyclosporine A on neurodegeneration and motor impairment in rotenone-induced experimental models of Parkinson's disease

The development of neuroprotective drugs targeting mitochondria could be an important strategy in combating the progressive clinical course of Parkinson's disease. In the current study, we demonstrated that in SH-SY5Y cells (human dopaminergic neuroblastoma cell line), rotenone caused a dose-dependent (0.25–1 μM) and time-dependent (up to 48 h) loss of cell viability and a loss of cellular ATP content with mitochondrial membrane depolarization and an increased formation of reactive oxygen species; all these processes were markedly prevented by the mitochondrial permeability transition pore blocker cyclosporine A, which did not affect complex I inhibition by rotenone. The nuclear morphology of rotenone-treated cells for 48 h indicated the presence of both necrosis and apoptosis. We then examined the effects of cyclosporine A on the rotenone-induced model of Parkinson's disease in Wistar rats. Cyclosporine A significantly improved the motor deficits and prevented the loss of nigral dopaminergic neurons projecting into the striatum in rotenone-treated rats. Being a marketed immuno-suppressive drug, cyclosporine A should be further evaluated for its putative neuroprotective action in Parkinson's disease.

The Antibody Dependant Neurite Outgrowth Modulation Response Involvement in Spinal Cord Injury.

Spinal cord injury (SCI) represents a major medical challenge. At present, there is still no cure to treat it efficiently and enable functional recovery below the injury site. Previously, we demonstrated that inflammation determines the fate of the physiopathology. To decipher the molecular mechanisms involved in this process, we performed a meta-analysis of our spatio-temporal proteomic studies in the time course of SCI. This highlighted the presence of IgG isotypes in both spinal cord explants and their secretomes. These IgGs were detected in the spinal cord even if no SCI occurred. However, during the time course following SCI, abundance of IgG1 and IgG2 subclasses (a, b, c) varied according to the spatial repartition. IgG1 was clearly mostly abundant at 12 h, and a switch to IgG2a was observed after 24 h. This IgG stayed predominant 3, 7, and 10 days after SCI. A protein related to IgM as well as a variable heavy chain were only detected 12 h after lesion. Interestingly, treatment with RhoA inhibitor influenced the abundance of the various IgG isotypes and a preferential switch to IgG2c was observed. By data reuse of rat dorsal root ganglion (DRG) neurons RNAseq datasets and RT-PCR experiments performed on cDNA from DRG sensory neurons ND7/23 and N27 dopaminergic neural cell lines, we confirmed expression of immunoglobulin heavy and light chains (constant and variable) encoding genes in neurons. We then identified CD16 and CD32b as their specific receptors in sensory neuron cell line ND7/23 and their activation regulated neurites outgrowth. These results suggest that during SCI, neuronal IgG isotypes are released to modulate neurites outgrowth. Therefore, we propose a new view of the SCI response involving an antibody dependent neurite outgrowth modulation (ADNM) which could be a precursor to the neuroinflammatory response in pathological conditions.

A Pretreatment with Isoorientin Attenuates Redox Disruption, Mitochondrial Impairment, and Inflammation Caused by Chlorpyrifos in a Dopaminergic Cell Line: Involvement of the Nrf2/HO-1 Axis.

The C-glucosyl flavone isoorientin (ISO) is obtained by humans from the diet and exhibits several cytoprotective effects, as demonstrated in different experimental models. However, it was not previously shown whether ISO would be able to prevent mitochondrial impairment in cells exposed to a chemical stressor. Thus, we treated the human neuroblastoma SH-SY5Y cells with ISO (0.5-20µM) for 18h before a challenge with chlorpyrifos (CPF) at 100µM for additional 24h. We observed that ISO prevented the CPF-induced lipid peroxidation and protein carbonylation and nitration in the membranes of mitochondria extracted from CPF-treated cells. ISO also attenuated the CPF-elicited increase in the production of reactive species in this experimental model. Moreover, ISO prevented the CPF-induced disruption in the activity of components of the oxidative phosphorylation (OXPHOS) system in the SH-SY5Y cells. ISO also promoted an anti-inflammatory action in the cells exposed to CPF. CPF caused a decrease in the activity of the enzyme heme oxygenase-1 (HO-1), a cytoprotective agent. On the other hand, ISO upregulated HO-1 activity in SH-SY5Y cells. Inhibition of HO-1 by zinc protoporphyrin-IX (ZnPP-IX) suppressed the cytoprotection induced by ISO in the CPF-treated cells. Besides, silencing of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) abolished the ISO-induced HO-1 upregulation and mitochondrial benefits induced by this flavone on the CPF-challenged cells. Thus, ISO protected mitochondria of the CPF-treated cells by an Nrf2/HO-1-dependent fashion in the SH-SY5Y cells.

Insights into the Pharmacokinetics and In Vitro Cell-Based Studies of the Imidazoline I2 Receptor Ligand B06.

The impact of neurodegenerative diseases (ND) is becoming unbearable for humankind due to their vast prevalence and the lack of efficacious treatments. In this scenario, we focused on imidazoline I2 receptors (I2-IR) that are widely distributed in the brain and are altered in patients with brain disorders. We took the challenge of modulating I2-IR by developing structurally new molecules, in particular, a family of bicyclic α-iminophosphonates, endowed with high affinity and selectivity to these receptors. Treatment of two murine models, one for age-related cognitive decline and the other for Alzheimer’s disease (AD), with representative compound B06 ameliorated their cognitive impairment and improved their behavioural condition. Furthermore, B06 revealed beneficial in vitro ADME-Tox properties. The pharmacokinetics (PK) and metabolic profile are reported to de-risk B06 for progressing in the preclinical development. To further characterize the pharmacological properties of B06, we assessed its neuroprotective properties and beneficial effect in an in vitro model of Parkinson’s disease (PD). B06 rescued the human dopaminergic cell line SH-SY5Y from death after treatment with 6-hydroxydopamine (6-OHDA) and showed a crucial anti-inflammatory effect in a cellular model of neuroinflammation. This research reveals B06 as a putative candidate for advancing in the difficult path of drug discovery and supports the modulation of I2-IR as a fresh approach for the therapy of ND.

Alpha-synuclein negatively controls cell proliferation in dopaminergic neurons

As researchers grapple with the mechanisms and implications of alpha-synuclein (α-syn) in neuropathology, it is often forgotten that the function(s) of α-syn in healthy cells remain largely elusive. Previous work has relied on observing α-syn localization in the cell or using knockout mouse models. Here, we address the specific role of α-syn in human dopaminergic neurons by disrupting its gene (SNCA) in the human dopaminergic neuron cell line, LUHMES. SNCA-null cells were able to differentiate grossly normally and showed modest effects on gene expression. The effects on gene expression were monodirectional, resulting primarily in the significant decrease of expression for 401 genes, implicating them as direct, or indirect positive targets of α-syn. Gene ontological analysis of these genes showed enrichment in terms associated with proliferation, differentiation, and synapse activity. These results add to the tapestry of α-syn biological functions. Significance statementThe normal functions of α-syn have remained controversial, despite its clear importance in Parkinson's Disease pathology, where it accumulates in Lewy bodies and contributes to neurodegeneration. Its name implies synaptic and nuclear functions, but how it participates at these locations has not been resolved. Via knock-out experiments in dopaminergic neurons, we implicate α-syn as a functional participant in synapse activity and in proliferation/differentiation, the latter being novel and provide insight into α-syn's role in neuronal development.

Overexpression of SIRT3 Suppresses Oxidative Stress-induced Neurotoxicity and Mitochondrial Dysfunction in Dopaminergic Neuronal Cells.

Sirtuin 3 (SIRT3), a well-known mitochondrial deacetylase, is involved in mitochondrial function and metabolism under various stress conditions. In this study, we found that the expression of SIRT3 was markedly increased by oxidative stress in dopaminergic neuronal cells. In addition, SIRT3 overexpression enhanced mitochondrial activity in differentiated SH-SY5Y cells. We also showed that SIRT3 overexpression attenuated rotenone- or H2O2-induced toxicity in differentiated SH-SY5Y cells (human dopaminergic cell line). We further found that knockdown of SIRT3 enhanced rotenone- or H2O2-induced toxicity in differentiated SH-SY5Y cells. Moreover, overexpression of SIRT3 mitigated cell death caused by LPS/IFN-γ stimulation in astrocytes. We also found that the rotenone treatment increases the level of SIRT3 in Drosophila brain. We observed that downregulation of sirt2 (Drosophila homologue of SIRT3) significantly accelerated the rotenone-induced toxicity in flies. Taken together, these findings suggest that the overexpression of SIRT3 mitigates oxidative stress-induced cell death and mitochondrial dysfunction in dopaminergic neurons and astrocytes.

Identification by proximity labeling of novel lipidic and proteinaceous potential partners of the dopamine transporter

Dopamine (DA) transporters (DATs) are regulated by trafficking and modulatory processes that probably rely on stable and transient interactions with neighboring proteins and lipids. Using proximity-dependent biotin identification (BioID), we found novel potential partners for DAT, including several membrane proteins, such as the transmembrane chaperone 4F2hc, the proteolipid M6a and a potential membrane receptor for progesterone (PGRMC2). We also detected two cytoplasmic proteins: a component of the Cullin1-dependent ubiquitination machinery termed F-box/LRR-repeat protein 2 (FBXL2), and the enzyme inositol 5-phosphatase 2 (SHIP2). Immunoprecipitation (IP) and immunofluorescence studies confirmed either a physical association or a close spatial proximity between these proteins and DAT. M6a, SHIP2 and the Cullin1 system were shown to increase DAT activity in coexpression experiments, suggesting a functional role for their association. Deeper analysis revealed that M6a, which is enriched in neuronal protrusions (filopodia or dendritic spines), colocalized with DAT in these structures. In addition, the product of SHIP2 enzymatic activity (phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]) was tightly associated with DAT, as shown by co-IP and by colocalization of mCherry-DAT with a specific biosensor for this phospholipid. PI(3,4)P2 strongly stimulated transport activity in electrophysiological recordings, and conversely, inhibition of SHIP2 reduced DA uptake in several experimental systems including striatal synaptosomes and the dopaminergic cell line SH-SY5Y. In summary, here we report several potential new partners for DAT and a novel regulatory lipid, which may represent new pharmacological targets for DAT, a pivotal protein in dopaminergic function of the brain.

AMPK S-sulfuration contributes to H2S donors-induced AMPK phosphorylation and autophagy activation in dopaminergic cells

Hydrogen sulfide (H2S) serves as a neuromodulator and regulator of neuroinflammation. It is reported to be therapeutic for Parkinson's disease (PD) animal and cellular models. However, whether it affects α-synuclein accumulation in dopaminergic cells, the key pathological feature in PD, is poorly understood. In this study we reported that exogenous H2S donors NaHS and GYY4137 (GYY) enhanced the autophagy activity, as indicated by the increases of autophagy marker LC3-II expression and LC3 dots formation even during lysosome inhibition in dopaminergic cell lines and HEK293 cells. The enhancement of H2S donors on autophagic flux was mediated by adenosine 5′-monophosphate-activated protein kinase (AMPK)-dependent mammalian target of rapamycin (mTOR) inhibition, as H2S donors activated AMPK but reduced the mTOR activity and H2S donors-induced LC3-II increase was diminished by mTOR activator. Moreover, point mutation of Cys302 into alanine (C302A) in AMPKα2 subunit abolished the AMPK activation and mTOR inhibition, as well as autophagic flux increase elicited by NaHS. Interestingly, NaHS triggered AMPK S-sulfuration, which was not observed in AMPK C302A-transfected cells. Further, NaHS was able to attenuate α-synuclein accumulation in a cellular model induced by dopamine oxidized metabolite 3, 4-dihydroxyphenylacetaldehyde (DOPAL), and this effect was interfered by autophagy inhibitor wortmannin and also eliminated in AMPK Cys302A-transfected cells. In sum, the findings identified a role of Cys302 S-sulfuration in AMPK activation induced by exogenous H2S and demonstrated that H2S donors could enhance the autophagic flux via AMPK-mTOR signaling and thus reduce α-synuclein accumulation in vitro.

β-Cells Different Vulnerability to the Parkinsonian Neurotoxins Rotenone, 1-Methyl-4-phenylpyridinium (MPP+) and 6-Hydroxydopamine (6-OHDA).

Neurotoxins such as rotenone, 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) are well known for their high toxicity on dopaminergic neurons and are associated with Parkinson’s disease (PD) in murine models and humans. In addition, PD patients often have glucose intolerance and may develop type 2 diabetes (T2D), whereas T2D patients have higher risk of PD compared to general population. Based on these premises, we evaluated the toxicity of these three toxins on pancreatic β-cell lines (INS-1 832/13 and MIN6) and we showed that rotenone is the most potent for reducing β-cells viability and altering mitochondrial structure and bioenergetics in the low nanomolar range, similar to that found in dopaminergic cell lines. MPP+ and 6-OHDA show similar effects but at higher concentration. Importantly, rotenone-induced toxicity was counteracted by α-tocopherol and partially by metformin, which are endowed with strong antioxidative and cytoprotective properties. These data show similarities between dopaminergic neurons and β-cells in terms of vulnerability to toxins and pharmacological agents capable to protect both cell types.

Platelet-derived growth factor (PDGF)-BB protects dopaminergic neurons via activation of Akt/ERK/CREB pathways to upregulate tyrosine hydroxylase.

AimsThe neurotropic growth factor PDGF‐BB was shown to have vital neurorestorative functions in various animal models of Parkinson's disease (PD). Previous studies indicated that the regenerative property of PDGF‐BB contributes to the increased intensity of tyrosine hydroxylase (TH) fibers in vivo. However, whether PDGF‐BB directly modulates the expression of TH, and the underlying mechanism is still unknown. We will carefully examine this in our current study.MethodMPTP‐lesion mice received PDGF‐BB treatment via intracerebroventricular (i.c.v) administration, and the expression of TH in different brain regions was assessed by RT‐PCR, Western blot, and immunohistochemistry staining. The molecular mechanisms of PDGF‐BB‐mediated TH upregulation were examined by RT‐PCR, Western blot, ChIP assay, luciferase reporter assay, and immunocytochemistry.ResultsWe validated a reversal expression of TH in MPTP‐lesion mice upon i.c.v administration of PDGF‐BB for seven days. Similar effects of PDGF‐BB‐mediated TH upregulation were also observed in MPP+‐treated primary neuronal culture and dopaminergic neuronal cell line SH‐SY5Y cells. We next demonstrated that PDGF‐BB rapidly activated the pro‐survival PI3K/Akt and MAPK/ERK signaling pathways, as well as the downstream CREB in SH‐SY5Y cells. We further confirmed the significant induction of p‐CREB in PDGF‐BB‐treated animals in vivo. Using a genetic approach, we demonstrated that the transcription factor CREB is critical for PDGF‐BB‐mediated TH expression. The activation and nucleus translocation of CREB were promoted in PDGF‐BB‐treated SH‐SY5Y cells, and the enrichment of CREB on the promoter region of TH gene was also increased upon PDGF‐BB treatment.ConclusionOur data demonstrated that PDGF‐BB directly regulated the expression of TH via activating the downstream Akt/ERK/CREB signaling pathways. Our finding will further support the therapeutic potential of PDGF‐BB in PD, and provide the possibility that targeting PDGF signaling can be harnessed as an adjunctive therapy in PD in the future.

Astaxanthin prevents mitochondrial impairment in the dopaminergic SH-SY5Y cell line exposed to glutamate-mediated excitotoxicity: Role for the Nrf2/HO-1/CO-BR axis

Mitochondrial dysfunction has been viewed in several diseases, including neurological disorders. In the glutamate (GLU)-mediated excitotoxicity, it has been described mitochondrial impairment, disrupted redox environment, and increased rates of cell death in the affected brain areas. Astaxanthin (AST) is a potent antioxidant and anti-inflammatory xanthophyll that also promotes beneficial mitochondria-related effects in brain cells. However, it is not completely clear how AST would be able to promote mitochondrial protection in those cell types. Thus, we investigated here how AST would protect mitochondria in the dopaminergic SH-SY5Y cell line exposed to GLU. AST was administrated to the cells at 1–40 μM for 24 h prior to the exposure to GLU at 80 mM for additional 24 h. AST prevented the GLU-induced impairment in the activity of the Complexes I and V, the loss in mitochondrial membrane potential (MMP), and the decline in the synthesis of ATP. AST also induced an antioxidant effect in the membranes of mitochondria obtained from the GLU-treated SH-SY5Y cells. Inhibition of the enzyme heme oxygenase-1 (HO-1) or silencing of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) suppressed the AST-promoted cellular and mitochondrial protection. Either tricarbonyldichlororuthenium(II) dimer (CORM-2, a source of carbon monoxide - CO) or bilirubin (BR), that are products of the HO-1-biliverdin reductase (BVR) axis, blocked some of the effects caused by GLU in the SH-SY5Y cells. Overall, our data demonstrate that AST prevented mitochondrial dysfunction by a mechanism related to the Nrf2/HO-1 axis in GLU-challenged cells.

Inhibition of γH2AX, COX-2 and regulation of antioxidant enzymes in MPP+-exposed SH-SY5Y cells pre-treated with rutin.

Many plant-derived bioactive compounds such as rutin are reportedly effective in attenuating neuronal death in most neurodegenerative disorders. Parkinson's disease (PD) is characterized by the gradual degeneration of dopaminergic neurons in the substantia nigra of the midbrain, and has previously been modelled in-vitro through the specific neurotoxic activity of 1-methyl-4-phenylpyridinium (MPP+) on dopaminergic neurons. Rutin is a bioflavonoid with multiple pharmacological effects, and this study investigated the neuroprotective effects of rutin in the human dopaminergic SH-SY5Y cell line using the neurotoxin MPP+. SH-SY5Y cells pretreated with rutin, were exposed to MPP+ and evaluated for cell viability, nitric oxide (NO), reactive oxygen species (ROS) and antioxidant enzymes activities. In addition, western blot techniques were used to determine the protein expression levels of γH2AX and COX-2. Rutin significantly attenuated MPP+-induced loss of cell viability, mitigated ROS and NO production and inhibited the disruption of antioxidant enzymes activity. It was also observed that rutin significantly reduced protein expression levels of γH2AX and COX-2 in SH-SY5Y cells treated with MPP+. Taken together, findings from this study tend to suggest that rutin is a promising neuroprotective compound for the treatment of PD through its effects on some of the mechanisms that characterize this neurodegenerative disease.

Microglial MT1 activation inhibits LPS-induced neuroinflammation via regulation of metabolic reprogramming.

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. Although its pathogenesis remains unclear, a number of studies indicate that microglia‐mediated neuroinflammation makes a great contribution to the pathogenesis of PD. Melatonin receptor 1 (MT1) is widely expressed in glia cells and neurons in substantia nigra (SN). Neuronal MT1 is a neuroprotective factor, but it remains largely unknown whether dysfunction of microglial MT1 is involved in the PD pathogenesis. Here, we found that MT1 was reduced in microglia of SN in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced PD mouse model. Microglial MT1 activation dramatically inhibited lipopolysaccharide (LPS)‐induced neuroinflammation, whereas loss of microglial MT1 aggravated it. Metabolic reprogramming of microglia was found to contribute to the anti‐inflammatory effects of MT1 activation. LPS‐induced excessive aerobic glycolysis and impaired oxidative phosphorylation (OXPHOS) could be reversed by microglial MT1 activation. MT1 positively regulated pyruvate dehydrogenase alpha 1 (PDHA1) expression to enhance OXPHOS and suppress aerobic glycolysis. Furthermore, in LPS‐treated microglia, MT1 activation decreased the toxicity of conditioned media to the dopaminergic (DA) cell line MES23.5. Most importantly, the anti‐inflammatory effects of MT1 activation were observed in LPS‐stimulated mouse model. In general, our study demonstrates that MT1 activation inhibits LPS‐induced microglial activation through regulating its metabolic reprogramming, which provides a mechanistic insight for microglial MT1 in anti‐inflammation.

Camptothecin Regulates Microglia Polarization and Exerts Neuroprotective Effects via Activating AKT/Nrf2/HO-1 and Inhibiting NF-κB Pathways In Vivo and In Vitro.

Microglia, the main immune cells in the brain, participate in the innate immune response in the central nervous system (CNS). Studies have shown that microglia can be polarized into pro-inflammatory M1 and anti-inflammatory M2 phenotypes. Accumulated evidence suggests that over-activated M1 microglia release pro-inflammatory mediators that damage neurons and lead to Parkinson’s disease (PD). In contrast, M2 microglia release neuroprotective factors and exert the effects of neuroprotection. Camptothecin (CPT), an extract of the plant Camptotheca acuminate, has been reported to have anti-inflammation and antitumor effects. However, the effect of CPT on microglia polarization and microglia-mediated inflammation responses has not been reported. In our study we found that CPT improved motor performance of mice and reduced the loss of neurons in the substantia nigra (SN) of the midbrain in LPS-injected mice. In the mechanism study, we found that CPT inhibited M1 polarization of microglia and promotes M2 polarization via the AKT/Nrf2/HO-1 and NF-κB signals. Furthermore, CPT protected the neuroblastoma cell line SH-SY5Y and dopaminergic neuron cell line MN9D from damage mediated by microglia activation. In conclusion, our results demonstrate that CPT regulates the microglia polarization phenotype via activating AKT/Nrf2/HO-1 and inhibiting NF-κB pathways, inhibits neuro-inflammatory responses, and exerts neuroprotective effects in vivo and in vitro.

Alpha-lipoic acid protects human dopaminergic neuronal cells against hydrogen peroxide-induced cell injury by inhibiting autophagy and apoptosis

Alpha-lipoic acid (ALA) is a naturally occurring antioxidant and has been previously used to treat diabetes and cardiovascular disease. However, the autophagy effects of ALA against oxidative stress-induced dopaminergic neuronal cell injury remain unclear. The aim of this study was to investigate the role of ALA in autophagy and apoptosis against oxidative stress in the SH-SY5Y human dopaminergic neuronal cell line. We examined SH-SY5Y phenotypes using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (cell viability/proliferation), 4′,6-diamidino-2-phenylindole dihydrochloride nuclear staining, Live/Dead cell assay, cellular reactive oxygen species (ROS) assay, immunoblotting, and immunocytochemistry. Our data showed ALA attenuated hydrogen peroxide (H2O2)-induced ROS generation and cell death. ALA effectively suppressed Bax up-regulation and Bcl-2 and BclxL down-regulation. Furthermore, ALA increased the expression of the antioxidant enzyme, heme oxygenase-1. Moreover, the expression of Beclin-1 and LC-3 autophagy biomarkers was decreased by ALA in our cell model. Combined, these data suggest ALA protects human dopaminergic neuronal cells against H2O2-induced cell injury by inhibiting autophagy and apoptosis.

On chip detection of glial cell-derived neurotrophic factor secreted from dopaminergic cells under magnetic stimulation

Glial cell-derived neurotrophic factor (GDNF) is a small protein potently promoting the survival of dopaminergic and motor neurons. GDNF can be secreted from different types of cells including the dopaminergic neural cell line, N27. N27 cells, a rat dopaminergic neural cell line, is regarded as a suitable in vitro model for Parkinson's disease (PD) research. For PD treatment, transcranial magnetic stimulation (TMS), a noninvasive therapeutic method, showed beneficial clinical effects, but the mechanism for its benefit is not understood. Because GDNF is a potent neurotrophic factor, it is of great value to evaluate if GDNF secretion from N27 cells can be affected by magnetic stimulation (MS). However, the current methods for detecting GDNF are time-consuming and expensive. In this paper we outline the detection of GDNF secretion from N27 cells by ultrasensitive nanopore thin film sensors (nanosensor) for the first time. As low as 2 pg/mL GDNF can be readily detected by the nanosensor. Furthermore, we show that MS can promote GDNF secretion from N27 cells. Specifically, the GDNF concentration in N27 cell-conditioned media under MS treatment shows statistically significant increase up to 2-fold after 5 days in vitro in comparison with the control. This nanosensor along with the in vitro PD model N27 cells provides a low-cost, easy-to-use, sensitive approach for studying potential cell biological mechanisms of the clinical benefits of MS on PD.

S-(+)-Pentedrone and R-(+)-methylone as the most oxidative and cytotoxic enantiomers to dopaminergic SH-SY5Y cells: Role of MRP1 and P-gp in cathinones enantioselectivity

Cathinone derivatives are the most representative group within new drugs market, which have been described as neurotoxic. Since cathinones, as pentedrone and methylone, are available as racemates, it is our aim to study the neuronal cytotoxicity induced by each enantiomer. Therefore, a dopaminergic SH-SY5Y cell line was used to evaluate the hypothesis of enantioselectivity of pentedrone and methylone enantiomers on cytotoxicity, oxidative stress, and membrane efflux transport (confirmed by in silico studies). Our study demonstrated enantioselectivity of these cathinones, being the S-(+)-pentedrone and R-(+)-methylone the most oxidative enantiomers and also the most cytotoxic, suggesting the oxidative stress as main cytotoxic mechanism, as previously described in in vitro studies. Additionally, the efflux transporter multidrug resistance associated protein 1 (MRP1) seems to play, together with GSH, a selective protective role against the cytotoxicity induced by R-(−)-pentedrone enantiomer. It was also observed an enantioselectivity in the binding to P-glycoprotein (P-gp), another efflux protein, being the R-(−)-pentedrone and S-(−)-methylone the most transported enantiomeric compounds. These results were confirmed, in silico, by docking studies, revealing that R-(−)-pentedrone is the enantiomer with highest affinity to MRP1 and S-(−)-methylone and R-(−)-pentedrone are the enantiomers with highest affinity to P-gp. In conclusion, our data demonstrated that pentedrone and methylone present enantioselectivity in their cytotoxicity, which seems to involve different oxidative reactivity as well as different affinity to the P-gp and MRP1 that together with GSH play a protective role.

Ndfip1 Prevents Rotenone-Induced Neurotoxicity and Upregulation of α-Synuclein in SH-SY5Y Cells.

Nedd4 family interacting protein 1 (Ndfip1) is an adaptor of Nedd4-family ubiquitin ligases. Experimental results showed that Ndfip1 had a potential neuroprotective effect in neurology diseases. However, the neuroprotective effect and the underlying mechanisms of Ndfip1 in Parkinson's disease (PD) have not yet been fully elucidated. Therefore, in this study, we explored the neuroprotective effect of Ndfip1 against mitochondrial complex I inhibitor rotenone in a human dopaminergic neuroblastoma SH-SY5Y cell line and further elucidated its possible underlying mechanisms. Our results showed that rotenone could induce the up-regulation of α-synuclein (α-syn) in both mRNA and protein levels. The expression of Ndfip1 decreased at 24 h after rotenone treatment. Further study showed that high expression of Ndfip1 could protect SH-SY5Y cells against rotenone-induced neurotoxicity and antagonize the rotenone-induced increase in α-syn protein levels. In addition, high expression of Ndfip1 inhibited rotenone-induced increase in the protein levels of caspase-3 and decrease in tyrosine hydroxylase (TH). Further study showed that Ndfip1 did not affect the protein expression of iron regulatory protein 1 (IRP1), transferrin receptor 1 (TfR1), while antagonized the increase in protein levels of P62 and ferritin L caused by rotenone. Our findings provide specific identification of Ndfip1 proteins to inhibit the increase of α-syn in rotenone-induced SH-SY5Y cells. Ndfip1 might be a new theoretical drug target for the prevention and treatment of PD.